Non-hydrophobic compounds for use in treating metastasis and/or cartilage defect

ABSTRACT

The present invention is directed to compounds, tautomers, stereoisomers, and chemically modified compounds thereof, and their use in preventing and/or treating tumors of metastasis and/or cartilage defect, and to a pharmaceutical composition comprising such compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. § 371 of International Patent Application PCT/EP2016/061659, filed May 24, 2016, designating the United States of America and published in English as International Patent Publication WO 2016/198256 A1 on Dec. 15, 2016, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 15171966.3, filed Jun. 12, 2015.

The present invention refers to non-hydrophobic compounds, their use in prevention and/or treatment of metastasis and/or cartilage defect, and to a pharmaceutical composition comprising such compound, wherein the compound or composition is for example administered orally.

TECHNICAL BACKGROUND

Malignant melanoma is the skin cancer with the highest mortality rate. It is characterized by an early onset of metastasis formation and rapid disease progression; in the case of systemic metastases the 5-year survival rate is less than 10% (Balch, C. M., et al., Prognostic Factors Analysis of 17,600 Melanoma Patients: Validation of the American Joint Committee on Cancer Melanoma Staging System. Journal of Clinical Oncology, 2001. 19(16): p. 3622-3634). In the TopS-EU-countries (Germany, UK, France, Spain, Italy), USA and Australia 125.000 incidences of primary tumors have been diagnosed in the year 2010; until 2025 an increase of 150.000 is expected (Globocan 2008). With the incidence of this malignancy increasing, new therapeutic attempts are simultaneously emerging. They include the BRAF V600E inhibitor Vemurafenib, the c-Kit inhibitor Imatinib, the anti-CTLA-4 antibody Ipilimumab, which activates the immune system, as well as anti-PD1 immune checkpoint inhibitors Nivolumab and Lambrolizumab; however, overall survival remains poor (McArthur, G. A., et al., Safety and efficacy of vemurafenib in BRAFV600E and BRAFV600K mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study. The Lancet Oncology, 2014. 15(3): p. 323-332).

MIA, an 11 kDa protein secreted by melanocytic tumor cells, has been linked to cellular migration and invasion, which leads to the formation of metastases. It has also been associated with immunosuppression of malignant melanoma (Jachimczak, P., et al., Inhibition of immunosuppressive effects of melanoma-inhibiting activity (MIA) by antisense techniques. International Journal of Cancer, 2005. 113(1): p. 88-92) and is a diagnostic serum marker for melanoma disease progression as it is barely expressed in healthy tissue except by differentiating chondrocytes. MIA, also known as CD-RAP (cartilage-derived retinoic acid-sensitive protein), is not only expressed in melanoma cells but also in chondrocytes and modulates regeneration of regeneration of defect cartilage (Schmid R, Schiffner S, Opolka A, Grässel S, Schubert T, Moser M, Bosserhoff A K. Cell Death Dis. 2010 Nov. 11). Analysis of the MIA/CD-RAP-knockout-mouse revealed that MIA/CD-RAP influences interactions between chondrocytes and their surrounding extracellular matrix, inhibits the proliferation of mesenchymal cells and promotes chondrocyte differentiation MIA is not only expressed in the context of melanoma but in numerous types of tumors, often late stage tumors such as breast cancer, glioma, pancreas carcinoma, and colon carcinoma etc. MIA allows the release of cells from the tumor by masking of binding sites for fibronectin and/or integrin and their migration in one direction which results in the invasion into other tissues and the formation of metastasis.

In WO 2011/113604 it is disclosed that MIA is functionally active as a homodimeric species and that peptides, e.g., AR71, are able to block the MIA-MIA interaction and in consequence to inhibit the formation of metastases and/or influence, cartilage formation, e.g., chondrocyte differentiation. No adverse effects were observed after a peptide treatment of mice. However, peptides generally make for poor drug candidates as they are readily degraded by proteases in the digestive tract or in the serum and are therefore typically not orally available.

Hence, the present invention is dirceted to improved inhibitors of the MIA dimerization for effective use in the prevention and/or treatment of metastasis and/or cartilage defect, which are for example even orally administrable.

SUMMARY

In the present invention compounds have been investigated which are non-hydrophobic and interact with the dimerization site of MIA comprising or formed by for example at least three amino acid residues of the MIA protein selected from cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103, cysteine 106, valine 64, tyrosine 69, aspartate 87, lysine 91, glycine 54, leucine 58, phenylalanine 59, alanine 7, lysine 53, arginine 55, arginine 57, arginine 85 and lysine 94. Alternatively the dimerization site is selected from cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103, cysteine 106, alanine 7, lysine 53, arginine 55, arginine 57, arginine 85, and lysine 94. A further selection is for example cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103 and cysteine 106.

The non-hydrophobic compounds are used alone or in combination for use in preventing and/or treating metastasis caused by the dimerization of MIA protein and/or a cartilage defect, wherein regeneration is inhibited by MIA dimerization, wherein the compound(s) is/are selected from the group consisting of compound 1 to 270 of Table 1, tautomers, stereoisomers as well as chemically modified compounds thereof.

The metastasis preventable and/or treatable by the compounds of the present invention is based on any primary tumor expressing MIA such as melanoma, breast cancer, glioma, pancreas carcinoma, colon carcinoma, etc. The metastasis is located for example in the liver, lung, bone, colon, stomach, nerves, lymph nodes, skin and/or brain.

The compound of the present invention is administered alone or in combination with other non-hydrophobic compounds of the present invention, and in combination with a chemotherapeutic (e.g., Vermurafenib, Ipilimumab, Trametinib, Dabradenib, Dacarbazine, Paclitaxel, Carboplatin, Interferon-alpha, Aldesleukin etc.) comprising for example also an inhibitory cytokine (e.g., TGF alpha, TGF beta, interleukin etc.).

The compounds are either administered at the same time or consecutively.

The present invention is further directed to a pharmaceutical composition comprising one or more compounds of the present invention for use in preventing and/or treating of metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein and/or a cartilage defect, wherein regeneration is inhibited by MIA dimerization, wherein the pharmaceutical composition comprises at least one compound selected from the group consisting of compound 1 to 270 of Table 1, tautomers, stereoisomers and chemically modified compounds thereof, and a pharmaceutically acceptable carrier and/or solvent and optionally a chemotherapeutic.

In one embodiment the compound or the pharmaceutical composition of the present invention is administered orally.

FIG. 1 depicts a model of a MIA dimer wherein compounds of the present invention are bound to the MIA-MIA interaction site, also called MIA dimerization site.

FIG. 2 shows predicted passing of the blood-brain-barrier (FIG. 2A), predicted oral availability (FIG. 2B) and predicted systemic toxicity (FIG. 2C) of compound 3 (F1811-0131).

FIG. 3 presents inhibition of MIA dimerization by compounds 1 to 6 measured in the HTFP assay.

FIG. 4 shows Boyden chamber assays demonstrating the inhibition of melanoma cell migration by compounds 1, 2 and 3, respectively.

FIG. 5 depicts compounds 1 to 6 tested on proliferation and attachment of fibroblasts (FIG. 5A, 5B) and kidney cells HEK-293 (FIG. 5C, 5D), respectively. None of the tested compounds has an effect on these cells.

FIG. 6 presents the inhibition of the proliferation of the melanoma cell Mel-Im line by compound 1.

DETAILED DESCRIPTION

As a general treatment concept, the selective inhibition of MIA dimerization is an attractive therapy concept since, apart from its expression in malignant melanoma, MIA is only expressed by differentiating chondrocytes. Furthermore, the inhibition strategy of the present invention targets secreted extracellular MIA; thus the need for cell permeability of the compounds of the present invention is avoided. The compounds of the present invention are specifically developed and used for preventing and/or treating metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein or a cartilage defect which results for example in mechanical or immunological destruction and suppression of regeneration of the cartilage by MIA dimerization. MIA avoids the dedifferentiation of chondrocytes which would allow the proliferation of the chondrocytes. Without (dimerized) MIA, the chondrocytes transform into a certain type of precursor cells which are ale to grow and to regenerate, i.e., close a cartilage defect. MIA blocks this regeneration.

The compounds of the present invention interact and bind respectively, with/to the dimerization site of MIA proteins. Thus, the compounds of the present invention avoid the dimerization of MIA proteins and/or break up existing MIA dimers.

The MIA dimerization site which is the site where MIA proteins interact comprises or is formed by at least three amino acid residues of said MIA protein selected from cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103, cysteine 106, valine 64, tyrosine 69, aspartate 87, lysine 91, glycine 54, leucine 58, phenylalanine 59, alanine 7, lysine 53, arginine 55, arginine 57, arginine 85 and lysine 94. In one embodiment, the dimerization site of a MIA protein comprises or consists of cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103, cysteine 106, alanine 7, lysine 53, arginine 55, arginine 57, arginine 85, and lysine 94. In another embodiment, the MIA dimerization site comprises or consists of cysteine 17, serine 18, tyrosine 47, glycine 61, glycine 66, aspartate 67, leucine 76, tryptophan 102, aspartate 103 and cysteine 106.

Via the dimerization site even more than two MIA proteins may interact and form an aggregate comprising or consisting of three or more MIA proteins. FIG. 1 presents a model of such MIA dimerization site virtually bound to numerous compounds of the present invention. The amino acids of the MIA protein forming the dimerization site are hydrophobic in that the dimerization site is hydrophobic. Surprisingly, the compounds of the present invention interacting with the MIA dimerization site are non-hydrophobic. The compounds of the present invention for use in preventing and/or treating metastasis caused by the dimerization of MIA protein or a cartilage defect are listed in the following Table 1. The invention further comprises the tautomers, stereoisomers and chemically modified compounds of the compounds listed in Table 1.

TABLE 1 Com- Reference pound of x Structure No. Tautomere/Stereoisomer Variant Com- pound 1

F3225- 8500

F3225- 8500_8_2 (1 of 28) Preferred IUPAC Name = 5-amino-4-(1H-1,3- benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro- 1H-pyrrol-3-one Com- pound 2

  Preferred IUPAC Name = N-[4-(1,2,3,4- tetrahydroisoquinolin-2-yl)but-2-yn-1- yl]methanesulfonamide F6244- 3097

F6244- 3097_1 (1 of 2) Com- pound 3

  Preferred IUPAC Name = 2-(2,6-dioxo-1,2,3,6- tetrahydropyrimidin-4-yl)-N-Pentylacetamide F1811- 0131

F1811- 0131_52_1 (1 of 76) Com- pound 4

  Preferred IUPAC Name = 1-{1-[2-hydroxy-3- (piperazin-1-yl)propyl]-2,4-dimethyl-1H- pyrrol-3-yl}ethan-1-one F3255- 0159

F3255- 0159_3_2 (1 of 8) Com- pound 5

F2145- 0409

F2145- 0409_5_1 (1 of 14) Preferred IUPAC Name = 5-methoxy-2- (piperazin-1-ylmethyl)-1H-1,3-Benzodiazole Com- pound 6

F2622- 0002

F2622- 0002_7_1 (1 of 40) Preferred IUPAC Name = N-butyl-2- hydroxy-9-methyl-4-oxo-4H- pyrido[1,2-a]pyrimidine-3-carboxamide Com- pound 7

F0050- 0006

F0050- 0006_2 (1 of 4) Com- pound 8

F0095- 1317

F0095- 1317_13_5 (1 of 116) Com- pound 9

F0126- 0203

F0126- 0203_16_4 (1 of 46) Com- pound 10

F0176- 0083

F0176- 0083_3_1 (1 of 10) Com- pound 11

F0196- 0408

F0196- 0408_1_1 (1 of 2) Com- pound 12

F0285- 0322

F0285- 0322_2 (1 of 2) Com- pound 13

F0347- 0341

F0347- 0341_21_1 (1 of 14) Com- pound 14

F0347- 0841

F0347- 0841_18_1 (1 of 12) Com- pound 15

F0578- 0190

F0578- 0190_7_1 (1 of 22) Com- pound 16

F0611- 0055

F0611- 0055_1 (1 of 4) Com- pound 17

F0693- 0722

F0693- 0722_14_1 (1 of 22) Com- pound 18

F0696- 0806

F0696- 0806_6_1 (1 of 44) Com- pound 19

F0696- 0849

F0696- 0849_24_1 (1 of 88) Com- pound 20

F0704- 0019

F0704- 0019_7_1 (1 of 11) Com- pound 21

F0719- 0066

F0719- 0066_1 (1 of 4) Com- pound 22

F0719- 0070

F0719- 0070_1_1 (1 of 4) Com- pound 23

F0719- 0145

F0719- 0145_1_1 (1 of 4) Com- pound 24

F0815- 0092

F0815- 0092_1_1 (1 of 7) Com- pound 25

F0840- 0033

F0840- 0033_2 (1 of 4) Com- pound 26

F0840- 0034

F0840- 0034_1_1 (1 of 4) Com- pound 27

F0840- 0058

F0840- 0058_1 (1 of 4) Com- pound 28

F0840- 0059

F0840- 0059_1 (1 of 8) Com- pound 29

F0840- 0204

F0840- 0204_1_2 (1 of 4) Com- pound 30

F0840- 0206

F0840- 0206_2 (1 of 4) Com- pound 31

F0840- 0214

F0840- 0214_2 (1 of 4) Com- pound 32

F0896- 0201

F0896- 0201_1_2 (1 of 4) Com- pound 33

F0910- 6535

F0910- 6535_23_1 (1 of 124) Com- pound 34

F0915- 2932

F0915- 2932_3_1 (1 of 8) Com- pound 35

F0915- 2972

F0915- 2972_3_1 (1 of 5) Com- pound 36

F0918- 1472

F0918- 1472_8_1 (1 of 10) Com- pound 37

F0919- 7794

F0919- 7794_104_3 (1 of 270) Com- pound 38

F1001- 0002

F1001- 0002_5_1 (1 of 12) Com- pound 39

F1016- 0141

F1016- 0141_1_1 (1 of 2) Com- pound 40

F1016- 0159

F1016- 0159_1_1 (1 of 2) Com- pound 41

F1021- 0224

F1021- 0224_1_2 (1 of 4) Com- pound 42

F1032- 0001

F1032- 0001_1_2 (1 of 4) Com- pound 43

F1032- 0021

F1032- 0021_1_2 (1 of 4) Com- pound 44

F1032- 0031

F1032- 0031_1 (1 of 4) Com- pound 45

F1065- 0751

F1065- 0751_3_1 (1 of 8) Com- pound 46

F1107- 0031

F1107- 0031_80_4 (1 of 448) Com- pound 47

F1126- 0569

F1126- 0569_4_1 (1 of 6) Com- pound 48

F1132- 0388

F1132- 0388_1_1 (1 of 1) Com- pound 49

F1174- 1011

F1174- 1011_1_1 (1 of 2) Com- pound 50

F1243- 0186

F1243- 0186_3_1 (1 of 6) Com- pound 51

F1360- 0381

F1360- 0381_3_1 (1 of 4) Com- pound 52

F1387- 0270

F1387- 0270_2_1 (1 of 3) Com- pound 53

F1411- 0031

F1411- 0031_9_1 (1 of 14) Com- pound 54

F1438- 0002

F1438- 0002_2 (1 of 4) Com- pound 55

F1438- 0006

F1438- 0006_1_3 (1 of 8) Com- pound 56

F1440- 0010

F1440- 0010_9_1 (1 of 14) Com- pound 57

F1478- 0152

F1478- 0152_1 (1 of 2) Com- pound 58

F1501- 0026

F1501- 0026_1 (1 of 4) Com- pound 59

F1533- 0056

F1533- 0056_2_2 (1 of 8) Com- pound 60

F1549- 0007

F1549- 0007_1_1 (1 of 4) Com- pound 61

F1562- 0050

F1562- 0050_1 (1 of 4) Com- pound 62

F1562- 0059

F1562- 0059_1_2 (1 of 4) Com- pound 63

F1605- 0391

F1605- 0391_4_1 (1 of 37) Com- pound 64

F1607- 0384

F1607- 0384_32_1 (1 of 66) Com- pound 65

F1607- 0387

F1607- 0387_33_1 (1 of 33) Com- pound 66

F1630- 0018

F1630- 0018_2 (1 of 4) Com- pound 67

F1681- 0025

F1681- 0025_1_2 (1 of 4) Com- pound 68

F1681- 0068

F1681- 0068_2 (1 of 4) Com- pound 69

F1681- 0091

F1681- 0091_1 (1 of 4) Com- pound 70

F1709- 0008

F1709- 0008_1_1 (1 of 4) Com- pound 71

F1723- 0297

F1723- 0297_2_2 (1 of 8) Com- pound 72

F1725- 0012

F1725- 0012_1_1 (1 of 4) Com- pound 73

F1771- 0001

F1771- 0001_2 (1 of 4) Com- pound 74

F1771- 0003

F1771- 0003_1_2 (1 of 4) Com- pound 75

F1771- 0007

F1771- 0007_1_8 (1 of 16) Com- pound 76

F1811- 0028

F1811- 0028_57_1 (1 of 139) Com- pound 77

F1811- 0068

F1811- 0068_142_1 (1 of 211) Com- pound 78

F1811- 0098

F1811- 0098_148_1 (1 of 238) Com- pound 79

F1811- 0101

F1811- 0101_142_1 (1 of 203) Com- pound 80

F1811- 0112

F1811- 0112_137_1 (1 of 192) Com- pound 81

F1811- 0121

F1811- 0121_142_1 (1 of 171) Com- pound 82

F1863- 0017

F1863- 0017_2 (1 of 28) Com- pound 83

F1894- 0028

F1894- 0028_3_1 (1 of 6) Com- pound 84

F1894- 0031

F1894- 0031_3_1 (1 of 3) Com- pound 85

F1894- 0157

F1894- 0157_3_1 (1 of 5) Com- pound 86

F1967- 1078

F1967- 1078_1 (1 of 4) Com- pound 87

F1967- 1118

F1967- 1118_22_1 (1 of 41) Com- pound 88

F1967- 1334

F1967- 1334_1_1 (1 of 2) Com- pound 89

F1984- 0137

F1984- 0137_3_1 (1 of 4) Com- pound 90

F1986- 0021

F1986- 0021_1 (1 of 4) Com- pound 91

F1986- 0024

F1986- 0024_1_1 (1 of 4) Com- pound 92

F2011- 0385

F2011- 0385_1 (1 of 2) Com- pound 93

F2101- 0054

F2101- 0054_8_1 (1 of 18) Com- pound 94

F2101- 0132

F2101- 0132_7_1 (1 of 18) Com- pound 95

F2137- 0016

F2137- 0016_1_1 (1 of 4) Com- pound 96

F2145- 0090

F2145- 0090_3_1 (1 of 4) Com- pound 97

F2145- 0296

F2145- 0296_3_1 (1 of 4) Com- pound 98

F2145- 0373

F2145- 0373_3_1 (1 of 7) Com- pound 99

F2145- 0423

F2145- 0423_5_1 (1 of 7) Com- pound 100

F2145- 0424

F2145- 0424_1_1 (1 of 7) Com- pound 102

F2145- 0478

F2145- 0478_2_1 (1 of 4) Com- pound 103

F2146- 0582

F2146- 0582_3_1 (1 of 4) Com- pound 104

F2147- 0176

F2147- 0176_1_1 (1 of 4) Com- pound 105

F2147- 0549

F2147- 0549_3_2 (1 of 8) Com- pound 106

F2148- 0005

F2148- 0005_6_4 (1 of 28) Com- pound 107

F2148- 0237

F2148- 0237_2_1 (1 of 7) Com- pound 108

F2150- 0003

F2150- 0003_3_1 (1 of 4) Com- pound 109

F2150- 0040

F2150- 0040_3_1 (1 of 4) Com- pound 110

F2158- 0133

F2158- 0133_1_1 (1 of 7) Com- pound 111

F2158- 0625

F2158- 0625_1 (1 of 7) Com- pound 112

F2158- 0833

F2158- 0833_3_1 (1 of 4) Com- pound 113

F2158- 1073

F2158- 1073_4_1 (1 of 7) Com- pound 114

F2164- 0018

F2164- 0018_5_1 (1 of 7) Com- pound 115

F2182- 0033

F2182- 0033_1_2 (1 of 4) Com- pound 116

F2182- 0073

F2182- 0073_3_1 (1 of 4) Com- pound 117

F2182- 0075

F2182- 0075_3_1 (1 of 4) Com- pound 118

F2182- 0085

F2182- 0085_1_1 (1 of 4) Com- pound 119

F2182- 0091

F2182- 0091_1_1 (1 of 4) Com- pound 120

F2182- 0097

F2182- 0097_1_1 (1 of 4) Com- pound 121

F2182- 0103

F2182- 0103_3_1 (1 of 4) Com- pound 122

F2182- 0106

F2182- 0106_3_1 (1 of 4) Com- pound 123

F2182- 0107

F2182- 0107_1_1 (1 of 4) Com- pound 124

F2182- 0112

F2182- 0112_1_1 (1 of 4) Com- pound 125

F2185- 0004

F2185- 0004_3_1 (1 of 4) Com- pound 126

F2185- 0006

F2185- 0006_1_1 (1 of 4) Com- pound 127

F2185- 0014

F2185- 0014_3_2 (1 of 14) Com- pound 128

F2189- 0334

F2189- 0334_5_2 (1 of 14) Com- pound 129

F2206- 0079

F2206- 0079_2_1 (1 of 3) Com- pound 130

F2274- 0585

F2274- 0585_3_1 (1 of 4) Com- pound 131

F2416- 0083

F2416- 0083_1 (1 of 1) Com- pound 132

F2503- 0105

F2503- 0105_22_1 (1 of 39) Com- pound 133

F2551- 0031

F2551- 0031_2_1 (1 of 18) Com- pound 134

F2622- 0072

F2622- 0072_41_1 (1 of 129) Com- pound 135

F2622- 0218

F2622- 0218_20_1 (1 of 40) Com- pound 136

F2624- 0002

F2624- 0002_7_1 (1 of 40) Com- pound 137

F2624- 0008

F2624- 0008_7_1 (1 of 40) Com- pound 138

F2643- 0132

F2643- 0132_2_1 (1 of 4) Com- pound 139

F2711- 1093

F2711- 1093_10_1 (1 of 60) Com- pound 140

F2713- 0139

F2713- 0139_4_1 (1 of 5) Com- pound 141

F2716- 0876

F2716- 0876_15_1 (1 of 14) Com- pound 142

F2716- 0877

F2716- 0877_6_1 (1 of 8) Com- pound 143

F2721- 0110

F2721- 0110_6_2 (1 of 14) Com- pound 144

F2811- 0081

F2811- 0081_150_4 (1 of 451) Com- pound 145

F2882- 0062

F2882- 0062_1_1 (1 of 21) Com- pound 146

F3012- 0022

F3012- 0022_1_2 (1 of 4) Com- pound 147

F3034- 0039

F3034- 0039_1 (1 of 8) Com- pound 148

F3034- 0286

F3034- 0286_3_2 (1 of 4) Com- pound 149

F3045- 0001

F3045- 0001_11_1 (1 of 14) Com- pound 150

F3084- 0058

F3084- 0058_2_1 (1 of 4) Com- pound 151

F3084- 0157

F3084- 0157_2_2 (1 of 8) Com- pound 152

F3099- 0269

F3099- 0269_1_1 (1 of 8) Com- pound 153

F3099- 3404

F3099- 3404_29_2 (1 of 120) Com- pound 154

F3099- 3503

F3099- 3503_1_2 (1 of 24) Com- pound 155

F3129- 0147

F3129- 0147_6_3 (1 of 24) Com- pound 156

F3146- 0231

F3146- 0231_2_1 (1 of 8) Com- pound 157

F3154- 0012

F3154- 0012_1 (1 of 2) Com- pound 158

F3168- 1703

F3168- 1703_19_2 (1 of 46) Com- pound 159

F3200- 0073

F3200- 0073_1_1 (1 of 16) Com- pound 160

F3200- 0074

F3200- 0074_1_1 (1 of 16) Com- pound 161

F3200- 0076

F3200- 0076_1_17 (1 of 53) Com- pound 162

F3219- 0003

F3219- 0003_23_1 (1 of 27) Com- pound 163

F3225- 8622

F3225- 8622_3_1 (1 of 4) Com- pound 164

F3230- 0054

F3230- 0054_1 (1 of 1) Com- pound 165

F3250- 0673

F3250- 0673_1 (1 of 4) Com- pound 166

F3250- 0686

F3250- 0686_2_2 (1 of 14) Com- pound 167

F3250- 0689

F3250- 0689_1 (1 of 4) Com- pound 168

F3250- 0696

F3250- 0696_1 (1 of 4) Com- pound 169

F3250- 0733

F3250- 0733_1_1 (1 of 4) Com- pound 170

F3254- 0053

F3254- 0053_1_1 (1 of 4) Com- pound 171

F3254- 0054

F3254- 0054_3 (1 of 8) Com- pound 172

F3254- 0055

F3254- 0055_1_3 (1 of 8) Com- pound 173

F3254- 0056

F3254- 0056_1 (1 of 4) Com- pound 174

F3254- 0059

F3254- 0059_1_1 (1 of 4) Com- pound 175

F3254- 0063

F3254- 0063_1 (1 of 4) Com- pound 176

F3254- 0064

F3254- 0064_1 (1 of 4) Com- pound 177

F3254- 0065

F3254- 0065_1_1 (1 of 4) Com- pound 178

F3254- 0073

F3254- 0073_1_1 (1 of 8) Com- pound 179

F3254- 0075

F3254- 0075_1 (1 of 4) Com- pound 180

F3254- 0098

F3254- 0098_1_2 (1 of 4) Com- pound 181

F3254- 0105

F3254- 0105_1_6 (1 of 16) Com- pound 182

F3255- 0157

F3255- 0157_1_1 (1 of 8) Com- pound 183

F3260- 0881

F3260- 0881_7_1 (1 of 13) Com- pound 184

F3266- 0086

F3266- 0086_1_1 (1 of 2) Com- pound 185

F3276- 0018

F3276- 0018_2 (1 of 4) Com- pound 186

F3276- 0031

F3276- 0031_2 (1 of 4) Com- pound 187

F3276- 0044

F3276- 0044_2 (1 of 4) Com- pound 188

F3277- 0798

F3277- 0798_1_1 (1 of 2) Com- pound 189

F3278- 0013

F3278- 0013_4 (1 of 8) Com- pound 190

F3284- 7619

F3284- 7619_2 (1 of 20) Com- pound 191

F3284- 7869

F3284- 7869_1_1 (1 of 2) Com- pound 192

F3301- 0129

F3301- 0129_1_2 (1 of 4) Com- pound 193

F3308- 0605

F3308- 0605_7_2 (1 of 16) Com- pound 194

F3308- 1206

F3308- 1206_1 (1 of 8) Com- pound 195

F3308- 2774

F3308- 2774_5_1 (1 of 14) Com- pound 196

F3308- 3248

F3308- 3248_6_1 (1 of 12) Com- pound 197

F3308- 3604

F3308- 3604_3_1 (1 of 14) Com- pound 198

F3314- 0051

F3314- 0051_1 (1 of 16) Com- pound 199

F3314- 0053

F3314- 0053_1_3 (1 of 8) Com- pound 200

F3314- 0059

F3314- 0059_2 (1 of 4) Com- pound 201

F3314- 0071

F3314- 0071_2 (1 of 4) Com- pound 202

F3316- 0057

F3316- 0057_1_2 (1 of 4) Com- pound 203

F3316- 0139

F3316- 0139_3_2 (1 of 8) Com- pound 204

F3316- 0145

F3316- 0145_1_2 (1 of 4) Com- pound 205

F3316- 0146

F3316- 0146_1_2 (1 of 4) Com- pound 206

F3316- 0166

F3316- 0166_1_1 (1 of 4) Com- pound 207

F3320- 0301

F3320- 0301_5_1 (1 of 7) Com- pound 208

F3331- 0647

F3331- 0647_1_1 (1 of 4) Com- pound 209

F3350- 0739

F3350- 0739_1_1 (1 of 4) Com- pound 210

F3358- 0351

F3358- 0351_18_2 (1 of 26) Com- pound 211

F3358- 0389

F3358- 0389_1_1 (1 of 8) Com- pound 212

F3368- 0119

F3368- 0119_1_1 (1 of 4) Com- pound 213

F3394- 1172

F3394- 1172_1_1 (1 of 5) Com- pound 214

F3394- 1274

F3394- 1274_4_1 (1 of 5) Com- pound 215

F3406- 8965

F3406- 8965_39_2 (1 of 158) Com- pound 216

F3407- 5137

F3407- 5137_3_1 (1 of 3) Com- pound 217

F5017- 0030

F5017- 0030_1 (1 of 2) Com- pound 218

F5017- 0031

F5017- 0031_1_1 (1 of 2) Com- pound 219

F5017- 0074

F5017- 0074_1 (1 of 2) Com- pound 220

F5017- 0076

F5017- 0076_1_1 (1 of 2) Com- pound 221

F5080- 0092

F5080- 0092_14_1 (1 of 4) Com- pound 222

F5096- 0076

F5096- 0076_3_1 (1 of 7) Com- pound 223

F5096- 0179

F5096- 0179_3_1 (1 of 7) Com- pound 224

F5302- 0092

F5302- 0092_1_1 (1 of 2) Com- pound 225

F5461- 1122

F5461- 1122_1_1 (1 of 4) Com- pound 226

F5668- 0082

F5668- 0082_1 (1 of 4) Com- pound 227

F5754- 0084

F5754- 0084_2_1 (1 of 4) Com- pound 228

F5791- 2428

F5791- 2428_5_1 (1 of 6) Com- pound 229

F5806- 0308

F5806- 0308_1_1 (1 of 2) Com- pound 230

F5828- 0220

F5828- 0220_3_2 (1 of 14) Com- pound 231

F5831- 3069

F5831- 3069_2_2 (1 of 8) Com- pound 232

F5831- 6204

F5831- 6204_1_1 (1 of 4) Com- pound 233

F5856- 0133

F5856- 0133_3_1 (1 of 2) Com- pound 234

F5857- 0026

F5857- 0026_1 (1 of 2) Com- pound 235

F5857- 0927

F5857- 0927_1 (1 of 2) Com- pound 236

F5857- 1145

F5857- 1145_1_1 (1 of 8) Com- pound 237

F5857- 1524

F5857- 1524_1_1 (1 of 8) Com- pound 238

F5857- 4935

F5857- 4935_1_2 (1 of 8) Com- pound 239

F5857- 5314

F5857- 5314_1_2 (1 of 8) Com- pound 240

F5857- 5693

F5857- 5693_1_1 (1 of 8) Com- pound 241

F5857- 5862

F5857- 5862_1 (1 of 2) Com- pound 242

F5857- 6072

F5857- 6072_1_1 (1 of 4) Com- pound 243

F5857- 6266

F5857- 6266_1 (1 of 1) Com- pound 244

F5857- 7563

F5857- 7563_1_2 (1 of 4) Com- pound 245

F5857- 7901

F5857- 7901_3_2 (1 of 8) Com- pound 246

F5857- 9048

F5857- 9048_1_2 (1 of 4) Com- pound 247

F5871- 2878

F5871- 2878_1 (1 of 2) Com- pound 248

F5871- 2883

F5871- 2883_1 (1 of 2) Com- pound 249

F5871- 2885

F5871- 2885_1_1 (1 of 2) Com- pound 250

F5871- 2898

F5871- 2898_1 (1 of 2) Com- pound 251

F5871- 3594

F5871- 3594_1_1 (1 of 2) Com- pound 252

F5871- 3603

F5871- 3603_2_1 (1 of 3) Com- pound 253

F5871- 3626

F5871- 3626_1_1 (1 of 4) Com- pound 254

F5871- 3665

F5871- 3665_3_1 (1 of 4) Com- pound 255

F5871- 4854

F5871- 4854_1 (1 of 2) Com- pound 256

F5882- 4189

F5882- 4189_1_1 (1 of 8) Com- pound 257

F5950- 0041

F5950- 0041_3_1 (1 of 4) Com- pound 258

F5959- 0024

F5959- 0024_1_1 (1 of 7) Com- pound 259

F6037- 0307

F6037- 0307_3_1 (1 of 4) Com- pound 260

F6064- 0318

F6064- 0318_3_1 (1 of 4) Com- pound 261

F6064- 0331

F6064- 0331_1_1 (1 of 4) Com- pound 262

F6089- 7863

F6089- 7863_1_1 (1 of 2) Com- pound 263

F6210- 1183

F6210- 1183_2 (1 of 2) Com- pound 264

F6223- 0039

F6223- 0039_1_1 (1 of 2) Com- pound 265

F6286- 0747

F6286- 0747_1 (1 of 3) Com- pound 266

F9995- 0220

F9995- 0220_22_1 (1 of 10) Com- CH₃OC(O)CH₂C(O)CH₂C(O)CH₃ F9999- pound 0001 267 Com- NH₂C(S)NH₂ F9999- pound 0002 268 Com- pound 269

F9999- 0003 Com- pound 270

F9999- 0004

Table 1 presents compounds of the present invention as well as examples of tautomers and stereoisomers, respectively, the number of potential tautomer or stereoisomers is indicated in brackets in column 5, “reference of variant”.

A chemical modification of compounds 1 to 270 is any modification that results in a compound characterized by a specific and effective interaction with or binding to the dimerization site of MIA. Chemical modifications are for example selected from the group consisting of H, alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl or alkyl, arylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, heteroaryalkyl, and alkoxyalkyl, each of which is optionally substituted one, two or three times with independently selected non-polar or polar groups.

“Alkyl” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. “Lower alkyl” as used herein, is a subset of alkyl, in some embodiments preferred, and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. The term “alkyl” or “loweralkyl” is intended to include both substituted and unsubstituted alkyl or loweralkyl unless otherwise indicated and these groups may be substituted with groups selected from halo (e.g., haloalkyl), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(0)m, haloalkyl-S(0)m, alkenyl-S(0)m, alkynyl-S(0)m, cycloalkyl-S(0)m, cycloalkylalkyl-S(0)m, aryl-S(0)m, arylalkyl-S(0)m, heterocyclo-S(0)m, heterocycloalkyl-S(0)m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxy acylamino, aminoacyloxy, nitro or cyano where m=0, 1, 2 or 3.

“Alkenyl” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in lower alkenyl 1 to 4 carbon atoms) which include 1 to 4 double bonds in the normal chain.

Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. The term “alkenyl” or “lower alkenyl” is intended to include both substituted and unsubstituted alkenyl or loweralkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and loweralkyl above.

“Alkynyl” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include 1 triple bond in the normal chain. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term “alkynyl” or “loweralkynyl” is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.

“Cycloalkyl” as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in a heterocyclic group as discussed below). Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. These rings may be optionally substituted with additional substituents as described herein such as halo or lower alkyl. The term “cycloalkyl” is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.

“Heterocyclic group” or “heterocyclo” as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or a bicyclic-ring system. Monocyclic ring systems are exemplified by any 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings include quaternized derivatives thereof and may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(O)m, haloalkyl-S(O)m, alkenyl-S(O)m, alkynyl-S(O)m, cycloalkyl-S(O)m, cycloalkylalkyl-S(O)m, aryl-S(O)m, arylalkyl-S(O)m, heterocyclo-S(O)m, heterocycloalkyl-S(O)m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m=0, 1, 2 or 3.

“Aryl” as used herein alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system. having one or more aromatic rings. Representative examples of aryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The term “aryl” is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.

“Arylalkyl” as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

“Heteroaryl” as used herein is as described in connection with heterocyclo above.

“Alkoxy” as used herein alone or as part of another group, refers to an alkyl or loweralkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -0-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

“Halo” as used herein refers to any suitable halogen, including —F, —CI, —Br, and —I.

Compound 1 is represented by the following general Formula (I)

wherein,

R₁, R₂ and R₄ represent, independent of each other, hydrogen, halogens, alkyls, alkenyls, alkynyls, cycloalkyls or aryls;

R₃ represents a linear n₁₋₇ alkyl, a branched n₃₋₇ alkyl, a linear n₁₋₇ alkenyl, a branched n₃₋₇ alkenyl, a linear n₁₋₇ alkynyl, a branched n₃₋₇ alkynyl, a n₅₋₇ cycloalkyl, a n₅₋₇ cycloalkenyl, a n₅₋₇ cycloalkynyl, a n₅₋₇ aryl;

X represents, independent of each other, a substituted or unsubstituted heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom;

Y represents a substituted or unsubstituted amine which may form a double bond with a neighboring carbon atom; and

Z represents a halogen or a substituted or unsubstituted heteroatom selected from O, N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom.

Compound 1 comprises for example the following modifications:

Compound 2 is represented by the following general Formula (II)

wherein,

R₁ represents a hydrogen, alkyl, alkenyl, alkynyl, amin, aminoalkyl, aminoalkenyl, halogens, alkyls, alkenyls, alkynyls, cycloalkyls or aryl;

R₂ represents, independent from each other, a hydrogen, alkyl, alkenyl, or alkynyl;

X represents a substituted or unsubstituted heteroatom selected from N and S; and

Y represents a linear n₃₋₅ alkyl, a linear n₃₋₅ alkenyl, or a linear n₃₋₅ alkynyl.

Compound 2 comprises for example the following modifications:

Compound 3 is represented by the following general Formula (III)

wherein,

R₁ represents a hydrogen, linear n₃₋₅ alkyl, a linear n₃₋₅ alkenyl, a linear n₃₋₅ alkynyl, a linear n₃₋₅ hydroxlylalkyl, a linear n₃₋₅ hydroxlylalkenyl, a linear n₃₋₅ hydroxlylalkynyl, a linear

n₃₋₅ ethoxylylalkyl, a linear n₃₋₅ ethoxylylalkenyl, or a linear n₃₋₅ ethoxylylalkynyl;

R₂ represents a hydrogen, halogen, alkyl, alkenyl, or alkynyl;

X represents a hydroxyl or a ═O group;

Y represents, independent of each other, a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom.

Compound 3 comprises for example the following modifications:

Compound 4 is represented by the following general Formula (IV)

wherein,

R₁ represents, independent from each other, a hydrogen, halogen, alkyl, alkenyl, or alkynyl;

R₂ represents, independent from each other, a hydrogen, halogen, alkyl, alkenyl, alkynyl or wherein the R₂ residues together form a n₅₋₇ cycloalkyl or an n₅₋₇ aryl;

X represents a hydrogen, a hydroxyl or a ═O group;

Y represents, independent from each other, a carbon atom or a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom.

Compound 4 comprises for example the following modifications:

Compound 5 is represented by the following general Formula (V)

wherein,

R₁ represents, independent of each other, a hydrogen, alkyl, alkenyl, or alkynyl, or wherein two R₁ form a cycloalkyl or an aryl;

R₂ represents, independent of each other, a hydrogen, alkyl, alkenyl, or alkynyl;

R₃ represents a hydrogen, halogen, ethoxyl, alkyl, alkenyl, or alkynyl;

Y represents, independent of each other, a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom; and

Z represents a carbon atom or N.

Compound 5 comprises for example the following modifications:

Compound 6 is represented by the following general Formula (VI)

wherein,

R₁ represents a linear n₂₋₆ alkyl, a branched n₃₋₆ alkyl, a linear n₂₋₆ alkenyl, a branched n₃₋₆ alkenyl, a linear n₂₋₆ alkynyl, a branched n₃₋₆ alkynyl, a n₅₋₇ cycloalkyl, a n₅₋₇ cycloalkenyl, a n₅₋₇ cycloalkynyl, a n₅₋₇ arylalkyl, a n₁₋₃ alkylcarboxyl;

R₂ represents, independent of each other, a hydrogen, alkyl, alkenyl, or alkynyl;

Y represents, independent of each other, a heteroatom selected from N and S, wherein the heteroatom may form a double bond with a neighboring carbon atom; and

Z represents, independent of each other, a carbon atom which may form a double bond with a neighboring carbon atom.

Compound 6 comprises for example the following modifications:

The present invention is not only directed to the non-hydrophobic compounds of Table 1, to the tautomers and stereoisomers thereof, but also to chemically modified forms of these compounds as mentioned for example above.

The metastasis preventable and/or treatable by use of a compound of the present invention is based on any type of primary tumor for example melanoma, breast cancer, glioma, pancreas carcinoma, and colon carcinoma. In one embodiment, the primary tumor is a late stage tumor. The metastasis is for example located in the liver, lung, bone, colon, stomach, nerves, lymph nodes, skin and/or brain.

The cartilage defect preventable and/or treatable by use of a compound of the present invention is for example a degenerative disorder of cartilage selected from rheumatoid arthritis, degeneration of cartilage in a joint, degenerative disc disease, meniscus tears, anterior crucial ligament (ACL) injury, arthritis, osteoarthritis, psoriatic arthritis, juvenile chronic arthritis, rhizomelic arthritis, rheumatoid poly-arthritis, synovitis and villonodular synovitis.

The dimerization of the MIA protein is for example measured by a heterogeneous transition metal-based fluorescence polarization (HTFP) assay, wherein the ratio P/P₀ is measured. P is the fluorescence polarization signal of a MIA protein labeled with a transition metal complex in the presence of substrate bound MIA-protein and of a compound to be tested. P₀ is the fluorescence polarization signal of free MIA-protein labeled with said luminescent transition metal complex in the absence of substrate bound MIA-protein and in the absence of the compound. In the absence of the compound, usually, the labeled MIA-protein would interact with the substrate bound MIA-protein, which, in turn, would contribute to a reduction in rotational mobility of the labeled MIA-protein, and therefore, the fluorescence polarization signal would increase upon such interaction. If, additionally, a compound is present that interferes with such interaction, no or little dimerization/aggregation occurs and no or little increase in fluorescence polarization signal would be detected. The smaller or even more negative P/P₀ is, the stronger such interference with dimer formation and aggregation is, and the better such compound prevents or breaks up dimerization/aggregation of MIA protein. In an alternative embodiment, binding of the compound to MIA protein is determined by NMR, e.g., heteronuclear NMR, e.g., ¹⁵N-¹H-HSQC-NMR.

In the present invention at least one compound of 1 to 270 of Table 1, a tautomer, a stereoisomer or a chemically modified compound thereof is administered to a subject, or in another embodiment two or more of these compounds, tautomers, stereoisomers and/or chemically modified compounds thereof are administered to a subject for use in preventing and/or treating of metastasis or a cartilage defect. The compounds are administered at the same time or consecutively.

In one embodiment the size of compounds of the present invention is increased by compound growing or compound linking. Regarding fragment growing an initial compound is steadily build up to explore favorable interactions with adjacent regions of the dimerization site; and regarding compound linking compounds of the present invention (e.g., two or more for example 3, 4, 5, 6, 7, 8, 9, 10) are connected to each other, i.e., are coupled with or without a linker.

In an embodiment of the present invention the compounds are hydrophobic, in another embodiment the compounds are hydrophilic or neutral.

In another embodiment one or more compounds of the present invention are administered to a subject in combination with a chemotherapeutic which is any chemical agent, e.g., naturally occurring or synthesized, effective in the treatment of cancer such as Vermurafenib, Ipilimumab, Trametinib, Dabradenib, Dacarbazine, Paclitaxel, Carboplatin, Interferon-alpha and Aldesleukin, TGF alpha, TGF beta, interleukin etc. The compound and the chemotherapeutic are administered at the same time or consecutively.

The present invention further refers to a pharmaceutical composition for use in preventing and/or treating of metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein and/or a cartilage defect, wherein regeneration is inhibited by MIA dimerization, wherein the pharmaceutical composition comprises at least one compound selected from the group consisting of compound 1 to 270 of Table 1, tautomers, stereoisomers and/or chemically modified compounds thereof, and a pharmaceutically acceptable carrier and/or solvent. The pharmaceutical composition may further comprise a chemotherapeutic or any substance, including cells such as chondrocytes, positively influencing cartilage growth and regeneration, respectively.

EXAMPLES

The following examples show the present invention in more detail, however, the invention is not limited to these examples.

Example 1

Fragment-based in silico screening was applied to identify the small, fragment-sized compounds of the present invention that inhibit MIA. The suggested structures were screened in vitro and modular synthesis strategies were developed for the most promising molecules.

NMR Titration Experiment

Derived from HSQC titrations of ¹⁵N-labeled MIA with AR71 (a peptide having the amino acid sequence FHWRYPLPLPGQ (SEQ ID NO:1)), the amino acids CYS17, SER18, TYR47, GLY66, ASP67, LEU76, TRP102, ASP103, and CYS106 were found to exhibit strong shift perturbations (Schmidt, J., A. Riechers, and A. K. Bosserhoff, MIA—a new target protein for malignant melanoma therapy. Histol Histopathol, 2013. 28(4): p. 421-6) and were defined as interacting residues in an in silico protein-peptide docking of MIA and AR71. The resulting model shows the binding of the peptide in the hydrophobic cleft, which forms part of the dimerization domain.

Virtual Screening

The 1.4 Å resolution crystal structure of human MIA protein 1I1J (Lougheed, J. C., et al., Structure of melanoma inhibitory activity protein, a member of a recently identified family of secreted proteins. Proceedings of the National Academy of Sciences, 2001, 98(10): p. 5515-5520) and the first model of each of the NMR solution structures 1K0X (Lougheed, J., P. Domaille, and T. Handel, Solution structure and dynamics of melanoma inhibitory activity protein. Journal of Biomolecular NMR, 2002, 22(3): p. 211-223) and 1HJD (Stoll, R., et al., The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold. 2001. 20(3): p. 340-349) were used as distinct MIA protein receptor conformations for the virtual screening experiment. The same amino acid residues that were used in the protein-peptide docking were provided as input to the molecular docking software in order to guide the automated detection and definition of putative binding sites in the MIA protein.

In each of the three MIA protein conformations the same single putative binding site was detected. The site is located in a cleft near the distal loop (residues 69 to 75) and is framed by the solvent exposed “disulfide loop” (residues 13 to 19), the turn of the RT loop (residues 35-38), and the C-terminal residues 102 to 106. Most of the amino acid residues identified to interact with the AR71 peptide by induced chemical shift changes constitute to the putative binding site, with the exception of TYR48, GLY66, and ASP67, which are located on the distal loop and thus too far away. Differences in the arrangement of the structural elements surrounding the cleft within each of the three distinct MIA receptor conformations result in a different volume and shape of the putative binding site.

Fragment library preparation commenced with 28751 fragment structures that were contained within the unprocessed fragment library catalogue obtained from the compound provider. Filtering for salts, unwanted chemistry and functional groups, as well as duplicate structures led to the removal of 1, 2577 and 127 structures, respectively. Each of the remaining 26046 structures was subjected to complete enumeration of its protonation and tautomeric states, which led to 87270 protomer variants for all fragment structures. Subsequently, all stereoisomers were enumerated for the protomer variants, which resulted in 124590 fragment variants. Generation of 3D conformations allowed for up to five alternate ring conformations per fragment variant and produced a final library of 217176 fragment variant conformations, which were docked into the three MIA receptor conformations with their respective putative binding sites.

The resulting docking poses were subjected to a filtering and ranking workflow, which, for each MIA protein conformation, determined a list of fragments ranked by the empirical scoring function of the docking software. The ten highest-ranking fragments for each MIA receptor conformation were selected for experimental testing.

All listed compounds that were screened for their ability to interfere with the MIA-MIA interaction (Table 1) in a heterogeneous transition metal-based fluorescence polarization (HTFP) assay previously developed (Riechers, A., et al., Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions. Biotechniques, 2009. 47(4): p. 837-44). This screening revealed significant decreases in the fluorescence polarization of Ru-(bpy)₃, labelled MIA for compounds 1, 2, 3, 4, 5, and 6; the results are shown in FIG. 3. The molecular structures of the compounds showing interference with the MIA-MIA interaction in the HTFP assay are shown in Table 1.

Example 2

Boyden chamber migration assays (Stoll, R., Lodermeyer, S. & Bosserhoff, A. K. Detailed analysis of MIA protein by mutagenesis. Biol Chem 387, 1601-1606, (2006)) using human Mel-Im melanoma cells demonstrated a reduction in the MIA activity on melanoma cell migration in the presence of compounds 1, 2, 3, 4, 5, and 6 at a concentration of 1 μM as shown in FIG. 4.

The melanoma cell line Mel-Im, established from a human metastatic bioptic sample (generous gift from Dr. Johnson, University of Munich, Germany) was used for the Boyden chamber migration experiments. All cells were maintained in DMEM (PAA, Pasching, Germany) supplemented with penicillin (400 U/mL), streptomycin (50 μg/mL), L-glutamine (300 μg/mL) and 10% fetal calf serum (Pan Biotech GmbH, Aidenbach, Germany) and split in a 1:6 ratio every three days. Migration assays were performed in Boyden Chambers containing polycarbonate filters with 8-nm pore size (Neuro Probe, Gaithersburg, Md., USA) essentially as described. MIA was added to the cell suspension at a final concentration of 200 ng/mL. Selected compounds were used at a final concentration of 1 μM. Experiments were carried out in triplicates and repeated at least three times.

Example 3

In order to assess whether compounds of the present invention show any adverse effects on normal cells, human fibroblasts and kidney cells were treated with the compounds 1, 2, 3, 4, 5, and 6, respectively, in vitro at a concentration of 7.8 μM in analogy to previous studies (Schmidt, J., et al., Targeting melanoma metastasis and immunosuppression with a new mode of melanoma inhibitory activity (MIA) protein inhibition. PLoS One, 2012. 7(5): p. e37941; Riechers, A., et al., Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions. Biotechniques, 2009. 47(4): p. 837-44). There were no negative effects on the proliferation of human fibroblasts (FIG. 5A) and kidney cells (FIG. 5C) as well as cellular attachment of the same cell types (FIGS. 5B and 5D) in the presence of the compounds.

A significant decrease in proliferation of the human melanoma cell line Mel Im after treatment with compound 1 was observed as presented in (FIG. 6). A phenomenon that could not be found with the other compounds 2, 3, 4, 5 or 6. 

The invention claimed is:
 1. A method of preventing the occurrence of and/or treating a subject for metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein or of a cartilage defect, wherein regeneration is inhibited by MIA dimerization, the method comprising: administering to the subject a composition comprising a compound that is non-hydrophobic, wherein the compound is selected from the group consisting of 5-amino-4-(1H-1,3-benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro-1H-pyrrol-3-one, N-[4-(1,2,3,4-tetrahydroisoquinolin-2-yl)but-2-yn-1-yl]methanesulfonamide, 2-(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N-pentylacetamide, 1-{1-[2-hydroxy-3-(piperazin-1-yl)propyl]-2,4-dimethyl-1H-pyrrol-3-yl}ethan-1-one, 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole, and N-butyl-2-hydroxy-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide.
 2. The method according to claim 1, wherein the compound is 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole.
 3. The method according to claim 1, wherein the metastasis is based on a primary tumor that is an MIA-expressing tumor.
 4. The method according to claim 3, wherein the primary tumor is selected from the group consisting of melanoma, breast cancer, glioma, pancreas carcinoma, and colon carcinoma.
 5. The method according to claim 1, wherein the metastasis is located in the liver, lung, bone, colon, stomach, nerves, lymph nodes, skin and/or brain.
 6. The method according to claim 1, wherein the composition is administered orally.
 7. The method according to claim 1, further comprising: administering a chemotherapeutic to the subject.
 8. The method according to claim 7, wherein the chemotherapeutic is selected from the group consisting of Vermurafenib, Ipilimumab, Trametinib, Dabradenib, Dacarbazine, Paclitaxel, Carboplatin, Interferon-alpha and Aldesleukin.
 9. The method according to claim 7, wherein the composition and the chemotherapeutic are administered at the same time.
 10. A method for preventing the occurrence of and/or treating a subject for a metastasis caused by the dimerization of melanoma inhibitory activity (MIA) protein and/or a cartilage defect, wherein regeneration is inhibited by MIA dimerization, the method comprising: administering to the subject a pharmaceutical composition comprising: at least one compound selected from the group consisting of 5-amino-4-(1H-1,3-benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro-1H-pyrrol-3-one, N-[4-(1,2,3,4-tetrahydroisoquinolin-2-yl)but-2-yn-1-yl]methanesulfonamide, 2-(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N-pentylacetamide, 1-{1-[2-hydroxy-3-(piperazin-1-yl)propyl]-2,4-dimethyl-1H-pyrrol-3-yl}ethan-1-one, 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole, and N-butyl-2-hydroxy-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide, and a pharmaceutically acceptable carrier and/or solvent.
 11. The method according to claim 10, further comprising: administering a chemotherapeutic to the subject.
 12. The method according to claim 10, wherein the compound is 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole.
 13. The method according to claim 7, wherein the chemotherapeutic is selected from the group consisting of vermurafenib, ipilimumab, trametinib, dabradenib, dacarbazine, paclitaxel, carboplatin, interferon-α, and aldesleukin.
 14. The method according to claim 10, wherein the pharmaceutical composition further comprises a chemotherapeutic.
 15. The method according to claim 7, wherein the composition and the chemotherapeutic are administered consecutively.
 16. The method according to claim 8, wherein the composition and the chemotherapeutic are administered at the same time.
 17. The method according to claim 8, wherein the composition and the chemotherapeutic are administered consecutively.
 18. A method of treating a subject in need thereof by inhibiting melanoma inhibitory activity protein (MIA)—MIA dimerization, the method comprising: administering to the subject a composition comprising a non-hydrophobic compound selected from the group consisting of 5-amino-4-(1H-1,3-benzodiazol-2-yl)-1-(butan-2-yl)-2,3-dihydro-1H-pyrrol-3-one, N-[4-(1,2,3,4-tetrahydroisoquinolin-2-yl)but-2-yn-1-yl]methanesulfonamide, 2-(2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)-N-pentylacetamide, 1-{1-[2-hydroxy-3-(piperazin-1-yl)propyl]-2,4-dimethyl-1H-pyrrol-3-yl}ethan-1-one, 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole, and N-butyl-2-hydroxy-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide.
 19. The method according to claim 18, wherein the subject has a primary tumor.
 20. The method according to claim 18, wherein the compound is administered orally.
 21. The method according to claim 18, wherein the compound is 5-methoxy-2-(piperazin-1-ylmethyl)-1H-1,3-benzodiazole. 